Hematopoietic stem cells are defined as clonogenic cells that can give rise to all blood cell lineages, as well as self-renew, at least for a significant period of time. There are 3 subsets of stem cells (HSC) and multipotent progenitors, and only the long-term (LT-HSC) self-renew for the life of the host, while ST-HSC renew for up to 8 weeks, and MPP for much less. Downstream of HSC/MPP are 2 classes of oligopotent progenitors - the common lymphoid progenitor (CLP) and the common myeloid progenitor (CMP). Each of these progenitors have been purified and can be prospectively identified by phenotype and isolated. In the first section of the grant, dividing LT-HSC, ST-HSC, and MPP will be tested for self-renewal or differentiation to one or the other downstream fates at the bulk level and at the single cell level to determine whether the cell fate decisions these multipotent cells make are largely symmetrical or asymmetrical. HSC cannot be expanded in vitro by any known protocol, but it is relatively simple to demonstrate their ability to expand by several orders of magnitude in various in vivo settings. To determine what intrinsic or extrinsic programs might regulate LT-HSC self-renewal, enforced expression of cell survival genes such as bcl-2, telomere extending genes such as TERT and TPC3, and genes downstream of particular signal transduction pathways such as beta catenin will be tested in HSC and other multipotent progenitors, and their progeny analyzed in an in vivo setting. Within the past year a large number of stem cells ranging from totipotent stem cells to organ specific stem cells (such as for the central nervous system) have been identified or claimed, with very surprising putative transdifferentiation possibilities between them and stem cells of the hematopoietic system. In this grant an effort will be made to establish unequivocal clonogenic assays for totipotent stem cells, and the relationship between various tissue specific stem cells, totipotent stem cells, and HSC will be examined. The study of HSC biology could be enhanced if HSC could be observed in situ by means of their expression of stem cell related genes. A long-term project that is part of this proposal is to begin to identify proteins that are useful fluorochromes among a plethora of recent discovered proteins such as the green fluorescent protein, and from amongst those that appear to be useful, to "knockin" different color genes as fusion proteins for these stem cell related genes already known (ckit, Sca1, Thy- 1.1, Flk2/Flt3, SCL) as well as others recently identified as being stem cell specific in their expression (e.g., a new serpin gene cloned in this laboratory).